Acidic residues in the purine binding site govern the 6-oxopurine specificity of the Leishmania donovani xanthine phosphoribosyltransferase

Buddy Ullman, Normand Cyr, Kenneth Choi, Armando Jardim

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Leishmania possess distinct xanthine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase enzymes that mediate purine salvage, an obligatory nutritional function for these pathogenic parasites. The xanthine phosphoribosyltransferase preferentially uses xanthine as a substrate, while the hypoxanthine-guanine phosphoribosyltransferase phosphoribosylates only hypoxanthine and guanine. These related phosphoribosyltransferases were used as model system to investigate the molecular determinants regulating the 6-oxopurine specificity of these enzymes. Analysis of the purine binding domains showed two conserved acidic amino acids; glutamate residues in the xanthine phosphoribosyltransferase (E198 and E215) and aspartate residues in the hypoxanthine-guanine phosphoribosyltransferase (D168 and D185). Genetic and biochemical analysis established that the single E198D and E215D mutations increased the turnover rates of the xanthine phosphoribosyltransferase without altering purine nucleobase specificity. However, the E215Q and E198,215D mutations converted the Leishmania xanthine phosphoribosyltransferase into a broad-specificity enzyme capable of utilizing guanine, hypoxanthine, and xanthine as substrates. Similarly, the D168,185E double mutation transformed the Leishmania hypoxanthine-guanine phosphoribosyltransferase into a mutant enzyme capable phosphoribosylating only xanthine, albeit with a much lower catalytic efficiency. These studies established that these conserved acidic residues play an important role in governing the nucleobase selectivity of the Leishmania 6-oxopurine phosphoribosyltransferases.

Original languageEnglish (US)
Pages (from-to)253-262
Number of pages10
JournalInternational Journal of Biochemistry and Cell Biology
Volume42
Issue number2
DOIs
StatePublished - Feb 2010

Fingerprint

Purinones
Hypoxanthine Phosphoribosyltransferase
Leishmania donovani
Leishmania
Xanthine
Binding Sites
Hypoxanthine
Guanine
Enzymes
Mutation
Acidic Amino Acids
Salvaging
Substrates
Aspartic Acid
Glutamic Acid
Molecular Biology
Parasites
purine
xanthine phosphoribosyltransferase

Keywords

  • HGPRT
  • Leishmania
  • Phosphoribosyltransferase
  • Purine metabolism
  • XPRT

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Cite this

Acidic residues in the purine binding site govern the 6-oxopurine specificity of the Leishmania donovani xanthine phosphoribosyltransferase. / Ullman, Buddy; Cyr, Normand; Choi, Kenneth; Jardim, Armando.

In: International Journal of Biochemistry and Cell Biology, Vol. 42, No. 2, 02.2010, p. 253-262.

Research output: Contribution to journalArticle

@article{c67b70ac591246e88e83cfc38b834b2e,
title = "Acidic residues in the purine binding site govern the 6-oxopurine specificity of the Leishmania donovani xanthine phosphoribosyltransferase",
abstract = "Leishmania possess distinct xanthine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase enzymes that mediate purine salvage, an obligatory nutritional function for these pathogenic parasites. The xanthine phosphoribosyltransferase preferentially uses xanthine as a substrate, while the hypoxanthine-guanine phosphoribosyltransferase phosphoribosylates only hypoxanthine and guanine. These related phosphoribosyltransferases were used as model system to investigate the molecular determinants regulating the 6-oxopurine specificity of these enzymes. Analysis of the purine binding domains showed two conserved acidic amino acids; glutamate residues in the xanthine phosphoribosyltransferase (E198 and E215) and aspartate residues in the hypoxanthine-guanine phosphoribosyltransferase (D168 and D185). Genetic and biochemical analysis established that the single E198D and E215D mutations increased the turnover rates of the xanthine phosphoribosyltransferase without altering purine nucleobase specificity. However, the E215Q and E198,215D mutations converted the Leishmania xanthine phosphoribosyltransferase into a broad-specificity enzyme capable of utilizing guanine, hypoxanthine, and xanthine as substrates. Similarly, the D168,185E double mutation transformed the Leishmania hypoxanthine-guanine phosphoribosyltransferase into a mutant enzyme capable phosphoribosylating only xanthine, albeit with a much lower catalytic efficiency. These studies established that these conserved acidic residues play an important role in governing the nucleobase selectivity of the Leishmania 6-oxopurine phosphoribosyltransferases.",
keywords = "HGPRT, Leishmania, Phosphoribosyltransferase, Purine metabolism, XPRT",
author = "Buddy Ullman and Normand Cyr and Kenneth Choi and Armando Jardim",
year = "2010",
month = "2",
doi = "10.1016/j.biocel.2009.10.020",
language = "English (US)",
volume = "42",
pages = "253--262",
journal = "International Journal of Biochemistry and Cell Biology",
issn = "1357-2725",
publisher = "Elsevier Limited",
number = "2",

}

TY - JOUR

T1 - Acidic residues in the purine binding site govern the 6-oxopurine specificity of the Leishmania donovani xanthine phosphoribosyltransferase

AU - Ullman, Buddy

AU - Cyr, Normand

AU - Choi, Kenneth

AU - Jardim, Armando

PY - 2010/2

Y1 - 2010/2

N2 - Leishmania possess distinct xanthine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase enzymes that mediate purine salvage, an obligatory nutritional function for these pathogenic parasites. The xanthine phosphoribosyltransferase preferentially uses xanthine as a substrate, while the hypoxanthine-guanine phosphoribosyltransferase phosphoribosylates only hypoxanthine and guanine. These related phosphoribosyltransferases were used as model system to investigate the molecular determinants regulating the 6-oxopurine specificity of these enzymes. Analysis of the purine binding domains showed two conserved acidic amino acids; glutamate residues in the xanthine phosphoribosyltransferase (E198 and E215) and aspartate residues in the hypoxanthine-guanine phosphoribosyltransferase (D168 and D185). Genetic and biochemical analysis established that the single E198D and E215D mutations increased the turnover rates of the xanthine phosphoribosyltransferase without altering purine nucleobase specificity. However, the E215Q and E198,215D mutations converted the Leishmania xanthine phosphoribosyltransferase into a broad-specificity enzyme capable of utilizing guanine, hypoxanthine, and xanthine as substrates. Similarly, the D168,185E double mutation transformed the Leishmania hypoxanthine-guanine phosphoribosyltransferase into a mutant enzyme capable phosphoribosylating only xanthine, albeit with a much lower catalytic efficiency. These studies established that these conserved acidic residues play an important role in governing the nucleobase selectivity of the Leishmania 6-oxopurine phosphoribosyltransferases.

AB - Leishmania possess distinct xanthine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase enzymes that mediate purine salvage, an obligatory nutritional function for these pathogenic parasites. The xanthine phosphoribosyltransferase preferentially uses xanthine as a substrate, while the hypoxanthine-guanine phosphoribosyltransferase phosphoribosylates only hypoxanthine and guanine. These related phosphoribosyltransferases were used as model system to investigate the molecular determinants regulating the 6-oxopurine specificity of these enzymes. Analysis of the purine binding domains showed two conserved acidic amino acids; glutamate residues in the xanthine phosphoribosyltransferase (E198 and E215) and aspartate residues in the hypoxanthine-guanine phosphoribosyltransferase (D168 and D185). Genetic and biochemical analysis established that the single E198D and E215D mutations increased the turnover rates of the xanthine phosphoribosyltransferase without altering purine nucleobase specificity. However, the E215Q and E198,215D mutations converted the Leishmania xanthine phosphoribosyltransferase into a broad-specificity enzyme capable of utilizing guanine, hypoxanthine, and xanthine as substrates. Similarly, the D168,185E double mutation transformed the Leishmania hypoxanthine-guanine phosphoribosyltransferase into a mutant enzyme capable phosphoribosylating only xanthine, albeit with a much lower catalytic efficiency. These studies established that these conserved acidic residues play an important role in governing the nucleobase selectivity of the Leishmania 6-oxopurine phosphoribosyltransferases.

KW - HGPRT

KW - Leishmania

KW - Phosphoribosyltransferase

KW - Purine metabolism

KW - XPRT

UR - http://www.scopus.com/inward/record.url?scp=73749085906&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=73749085906&partnerID=8YFLogxK

U2 - 10.1016/j.biocel.2009.10.020

DO - 10.1016/j.biocel.2009.10.020

M3 - Article

C2 - 19861168

AN - SCOPUS:73749085906

VL - 42

SP - 253

EP - 262

JO - International Journal of Biochemistry and Cell Biology

JF - International Journal of Biochemistry and Cell Biology

SN - 1357-2725

IS - 2

ER -